Non-invasive detection of pulmonary pathogens in ventilator-circuit filters by PCR

Abstract

Ventilator associated pneumonia is a common and costly complication in critically ill and injured surgical patients. The diagnosis of pneumonia remains problematic and non-specific. Using clinical criteria, a diagnosis of pneumonia is typically not made until an infection is well established. Semi-quantitative cultures of endotracheal aspirate and broncho-alveolar lavage are employed to improve the accuracy of diagnosis but are invasive and require time for culture results to become available. We report data that show that an inexpensive, rapid and non-invasive alternative may exist. In particular we show that: 1). Bio-aerosols evolved in the breath of ventilated patients and captured in the hygroscopic condenser humidifier filter of the ventilator circuit contain pathogenic micro-organisms. 2). The number (CFU/ml) and identity (Genus, species) of the pathogens in the aerosol samples can rapidly and inexpensively be determined by PCR. 3). Data from a convenience sample of filters correlate with clinical findings from standard microbiological methods such as broncho-alveolar lavage. The evaluation of the bacterial load evolved in exhaled breath by PCR is amenable to repeated sampling. Since increasing bacterial burden is believed to correlate with the establishment of infection, the use of quantitative PCR may provide a method to rapidly, inexpensively, and effectively detect and diagnose the early onset of pneumonia and identify pathogens involved.

Keywords: BAL; HCH; HME; Pneumonia; VAP; infection.

Figure 1

Depiction of the HCH filter in isolation and as part of the ventilator circuit. Panel a, at left, shows an example of the HCH filter unit that stands roughly two inches tall and less than an inch in diameter. The sponge can be seen at its center. Panel b, at center, shows a photograph of the HCH filter relative to the Y-valve and the endotracheal tube insertion point - blue cap. The schematic at right indicates the airflow and ancillary HEPA filters within the ventilator circuit.

Figure 2

A twelve hour time course of microbial growth at room temperature. One and 12 hour PIB (see methods) innoculated with E. coli fail to show growth relative to buffer alone (dark diamond). Error bars are derived from triplicate experiments. These data indicate that the HCH environment is bacteriostatic.

Figure 3

Time course of microbial viability of E. coli innoculated into one and 12 hour PIB as compared to viability in 150 mM NaCl buffer alone. Viability was determined by plate counts done in triplicate. The result of triplicate experiments indicate that the HCH environment is bacteriocidal.

Figure 4

Time course of quantitative microbial recovery from HCH filters innoculated with a prescribed initial number of E. coli. Cell numbers were determined using a Coulter counter as described in methods. The results of triplicate experiments show that percent recovery declines weakly with time but remains above 50%.

Figure 5

Standard curve relating the crossing threshold value from real time PCR with the number of E. coli cells assayed. The linear regression line has an r² value of 0.995. Similar standard curves are reported in [21].